The present invention relates generally to genetically engineered microorganisms and in particular to unique methods for stably incorporating exogenous DNA into cells, including the incorporation of multiple copies of the exogenous DNA at reiterated DNA sequences in the host. In a preferred aspect, the invention relates to yeasts capable of fermenting xylose (preferably cofermenting the same with glucose) to ethanol. More particularly, a preferred aspect of the invention relates to yeasts containing cloned genes encoding xylose reductase (XR), xylitol dehydrogenase (XD), and xylulokinase (XK), which yeasts substantially retain their efficiency for fermenting xylose to ethanol even after culturing in non-selective medium for a large number of generations.
As further background, recent studies have proven ethanol to be an ideal liquid fuel for automobiles. It can be used directly as a neat fuel (100% ethanol) or as a blend with gasoline at various concentrations. The use of ethanol to supplement or replace gasoline can reduce the dependency of many nations on imported foreign oil and also provide a renewable fuel for transportation. Furthermore, ethanol has proven to provide cleaner fuels that release far fewer pollutants into the environment than regular gasoline. For example, it has been demonstrated that the use of oxygenated materials in gasoline can reduce the emission of carbon monoxide, a harmful pollutant, into the air. Among the several oxygenates currently used for boosting the oxygen content of gasoline, ethanol has the highest oxygen content. The United States Environmental Protection Agency (EPA) has shown that gasoline blended with 10% ethanol reduces carbon monoxide emissions by about 25%-30%.
Up to now, the feedstock used for the production of industrial alcohol by fermentation has been sugars from sugar cane or beets and starch from corn or other food crops. However, these agricultural crops are presently considered to be too expensive to be used as feedstock for the large-scale production of fuel ethanol. Plant biomass is an attractive feedstock for ethanol-fuel production by fermentation because it is renewable, and available at low costs and in large amounts. The concept of using alcohol produced by microbial fermentation of sugars from agricultural biomass had its nascence at least two decades ago. The major fermentable sugars from cellulosic materials are glucose and xylose, with the ratio of glucose to xylose being approximately 2 or 3 to 1. The most desirable fermentations of cellulosic materials would, of course, completely convert both glucose and xylose to ethanol. Unfortunately, even now there is not a single known natural microorganism capable of fermenting both glucose and xylose effectively.
Yeasts, particularly Saccharomyces yeasts, have traditionally been used for fermenting glucose-based feedstocks to ethanol, and they are still considered the best microorganisms for that purpose. However, these glucose-fermenting yeasts, including the Saccharomyces yeasts, have been found to be unable to ferment xylose and also unable to use this pentose sugar for growth.
Recently, N. Ho et al. have developed recombinant yeasts, particularly recombinant Saccharomyces yeasts, capable of effectively fermenting xylose to ethanol (Ho and Tsao, 1995). More particularly, the preferred recombinant yeasts were capable of co-fermenting the two major sugar constituents of cellulosic biomass, glucose and xylose, to ethanol (Ho and Tsao, 1995). These recombinant yeasts were developed by the transformation of yeasts with a high-copy number plasmid containing three cloned genes, XR, XD, and XK, encoding three key enzymes for xylose metabolism (FIG. 1). FIG. 2 and FIG. 3 demonstrate two of the prior-made recombinant Saccharomyces yeasts, designated 1400(pLNH32) and 1400(pLNH33), capable of co-fermenting 8% glucose and 4% xylose present in the same medium almost completely to ethanol in two days. On the other hand, FIG. 4 shows that the parent yeast fusion 1400 (D'Amore, et al., 1989 and D'Amore, et al., 1990) can only ferment glucose, but not xylose, to ethanol. 1400(pLNH32) (in short LNH32) and 1400(pLNH33) (in short LNH33) were developed by the transformation of the Saccharomyces fusion 1400 (D'Amore, et al., 1989 and D'Amore, et al., 1990) with two of the high-copy-number plasmids, pLNH32 and pLNH33, shown in FIG. 1. To date, there have been four such high-copy-number plasmids reported, pLNH31, pLNH32, pLNH33, and pLNH34 (Ho and Tsao, 1995). Each of these plasmids can transform fusion 1400 to recombinant yeasts to co-ferment both glucose and xylose with similar efficiencies.
Yeasts 1400(pLNH32), 1400(pLNH33), and related recombinant xylose-fermenting Saccharomyces, with their xylose metabolizing genes cloned on a 2μ-based stable high-copy-number plasmid, are quite suitable for a batch process fermentation. However, in a continuous process fermentation, after prolonged culture in a glucose-rich medium (more than 20 generations), 1400(pLNH32), 1400(pLNH33), and similar plasmid-mediated recombinant yeasts lose their capability of fermenting xylose as shown in FIG. 5 and FIG. 6.
Generally, exogenous DNA or gene(s) can be cloned into yeasts by two separate ways. One way is to clone the exogenous DNA or gene(s) into a plasmid vector containing a selectable genetic marker and a functional yeast DNA replication origin or ARS (autonomous replicating sequence) (Struhl et al., 1979; Stinchcomb et al., 1980; Chan and Tye, 1980) that allows the plasmid to be able to replicate autonomously in its new host, followed by transformation of the desired yeast host with the plasmid containing the cloned DNA fragment or gene(s). The resulting yeast transformants are able to stably maintain the cloned gene in the presence of selection pressure. However, such cloned gene(s) are unstable after prolonged culture in non-selective medium (in the absence of selection pressure).
Another way to clone the exogenous DNA or gene(s) into a yeast host is to integrate the DNA or gene(s) into the yeast chromosome. In yeast, integrative transformation is almost always via homologous recombination (Orr-Weaver, 1981). The simplest way to clone a desired gene into a yeast chromosome by integration is first to clone the desired gene into a plasmid which does not contain a replication of origin or ARS (autonomous replication sequences) but does contain a piece of the host DNA for targeting the integration to a specific site (Orr-Weaver, 1981). Transformation of the new yeast host with such an intact integrative vector will generate integrative transformants containing the desired gene cloned to the site next to the selected targeting yeast DNA sequences. However, the frequency of such integrative transformation is extremely low (1 to 10 transformants per μg DNA). Subsequently, it has been demonstrated that integrative vectors linearized within the DNA fragment homologous to the host chromosomal DNA can transform yeasts with much higher frequencies (100- to 1000-fold higher) (Orr-Weaver, 1981; Orr-Weaver and Szostak, 1983). It was suggested that double-stranded breaks, introduced by restriction enzyme digestion, are recombinogenic and highly interactive with homologous chromosomal DNA. This is particularly helpful for a complex plasmid, containing more than one yeast gene, so that one can direct the integration to a specific site by making a restriction enzyme cut within the corresponding region on the plasmid.
Another type of integration, also described as transplacment or gene disruption, makes use of double homologous recombination to replace yeast chromosomal DNA (Rothstein, 1981). Double homologous recombination vectors contain the exogenous DNA or gene(s) to be cloned and the selection marker, flanked by yeast DNA sequences homologous to 5′ and 3′ regions of the segment of chromosomal DNA to be replaced. Prior to transformation, the vector is digested with restriction enzymes which liberate the transplacing fragment containing 5′ and 3′ ends homologous to the chromosomal DNA sequences at the desired integration sites. The latter strategy has become the method of choice for integrative transformation of yeast if a stable single-copy transformant is desired.
A number of strategies based on integration into reiterated chromosomal DNA have been used to generate stable multiple-copy integrants. For example, the delta sequence of yeast retrotransposon Ty (Sakai et al., 1990; Sakai et al., 1991), the highly conserved repeated sigma element (Kudla and Nicolas, 1992) and non-transcribed sequences of ribosomal DNA (Lopes et al., 1989; Lopes et al., 1991; Rossolini et al., 1992) have all been used as the target sites for multiple integration of exogenous gene(s) into yeast (Rothstein, 1991; Romanos et al., 1992).
Recent work reported in the literature on multiple integration of exogenous genes into the yeast chromosome has for the most part involved the use of either properly linearized non-replicative vectors or DNA fragments containing the desired gene(s) to be cloned and the genetic marker for selection, flanked with DNA sequences homologous to a region of yeast chromosomal DNA. Rarely, linearized replicative vectors and almost never intact replicative vectors, such as intact ARS vectors, were used to achieve such recombinant transformation. Thus, since early work at the onset of developing yeast integrative transformation, (Szoatak and Wu (1979)), and despite the observation that DNA cloned on ARS vectors can integrate into the host chromosomes (Cregg et al., 1985; Kurtz et al., 1986), the use of intact ARS vectors (Struhl et al., 1979; Stinchcomb et al., 1980; Chan and Tye, 1980) for integration purposes has long since generally been abandoned. This has especially been true since the discovery that the double-stranded breaks introduced by restriction enzyme digestion are recombinogenic (Orr-Weaver, 1981; Orr-Weaver and Szostak, 1983).
In light of this background, there remain needs for more stable yeast which ferment xylose to ethanol, preferably xylose and glucose simultaneously to ethanol, and for facile and effective methods for making high copy number integrants. The present invention addresses these needs.